Name: micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants
Uploaded: 2018-03-29T13:30:00
Description: Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

This work was funded by the University Paris-Diderot, the French National Institute of Health and Medical Research (INSERM), and the French Institute for Odontological Research (IFRO).

All animals used in the present study were maintained in accordance with guidelines for the care and use of laboratory animals from the French Ministry of Agriculture (A-75-06-12).
1. Animal Exposure to Toxicants
<ol>
	<li>Prior to performing this protocol, obtain necessary institutional approval and be sure to comply with all animal care guidelines.</li>
	<li>Apply the design of the research protocol allowing the constitution of different experimental groups in order to test the impact of t...

<ol>
	<li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel+defects+reflect+perinatal+exposure+to+bisphenol+A.">Enamel defects reflect perinatal exposure to bisphenol A.</a> Am J Pathol. 183, 108-118 (2013).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Exposure+to+Bisphenol+a+Exacerbates+Dental+Fluorosis+in+Growing+Rats.">Chronic Exposure to Bisphenol a Exacerbates Dental Fluorosis in Growing Rats.</a> J Bone Miner Res. 31, 1955-1966 (2016).</li><li>Alaluusua, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+dental+aberrations+after+the+dioxin+accident+in+Seveso.">Developmental dental aberrations after the dioxin accident in Seveso.</a> Environ Health Perspect. 112, 1313-1318 (2004).</li><li>Chapple, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+lifestyle,+behaviour+or+systemic+diseases+with+dental+caries+and+periodontal+diseases:+consensus+report+of+group+2+of+the+joint+EFP/ORCA+workshop+on+the+boundaries+between+caries+and+periodontal+diseases.">Interaction of lifestyle, behaviour or systemic diseases with dental caries and periodontal diseases: consensus report of group 2 of the joint EFP/ORCA workshop on the boundaries between caries and periodontal diseases.</a> J Clin Periodontol. 44, S39-S51 (2017).</li><li>Nanci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel:+Composition,+Formation,+and+Structure.">Enamel: Composition, Formation, and Structure.</a> Ten Cate's Oral Histology Development, Structure, and Function. , 122-164 (2012).</li><li>Leite, G. A., Sawan, R. M., Teofilo, J. M., Porto, I. M., Sousa, F. B., Gerlach, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+lead+exacerbates+dental+fluorosis.">Exposure to lead exacerbates dental fluorosis.</a> Arch Oral Biol. 56, 695-702 (2011).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel+hypomineralization+due+to+endocrine+disruptors.">Enamel hypomineralization due to endocrine disruptors.</a> Connect Tiss Res. 55, 1-5 (2014).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptor+involvement+in+rat+amelogenesis:+an+additional+way+for+endocrine+disrupting+chemicals+to+affect+enamel+synthesis.">Androgen receptor involvement in rat amelogenesis: an additional way for endocrine disrupting chemicals to affect enamel synthesis.</a> Endocrinology. 157, 4287-4296 (2016).</li><li>Weerheijm, K. L., Jalevik, B., Alaluusua, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molar-incisor+hypomineralisation.">Molar-incisor hypomineralisation.</a> Caries Res. 35, 390-391 (2001).</li><li>J&#228;levik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+Diagnosis+of+Molar-Incisor-+Hypomineralisation+(MIH):+A+systematic+review.">Prevalence and Diagnosis of Molar-Incisor- Hypomineralisation (MIH): A systematic review.</a> Eur Arch Paediatr Dent. 11, 59-64 (2010).</li><li>Alaluusua, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aetiology+of+Molar-Incisor+Hypomineralisation:+A+systematic+review.">Aetiology of Molar-Incisor Hypomineralisation: A systematic review.</a> Eur Arch Paediatr Dent. 11, 53-58 (2010).</li><li>Jedeon, K., Berdal, A., Babajko, S., Gibert, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tooth,+target+organ+of+Bisphenol+A,+could+be+used+as+a+biomarker+of+exposure+to+this+agent.">The tooth, target organ of Bisphenol A, could be used as a biomarker of exposure to this agent.</a> Bisphenol A: Sources, Risks of Environmental Exposure and Human Health Effects. , 205-225 (2015).</li><li>Fejerskov, O., Larsen, M. J., Richards, A., Baelum, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+tissue+effects+of+fluoride.">Dental tissue effects of fluoride.</a> Adv Dent Res. 8, 15-31 (1994).</li><li>Robinson, C., Connell, S., Kirkham, J., Brookes, S. J., Shore, R. C., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+fluoride+on+the+developing+tooth.">The effect of fluoride on the developing tooth.</a> Caries Res. 38, 268-276 (2004).</li><li>Sahlberg, C., Pavlic, A., Ess, A., Lukinmaa, P. 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J., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+32-kDa+fragment+the+functional+enamelin+unit+in+all+species?.">Is the 32-kDa fragment the functional enamelin unit in all species?.</a> Eur J Oral Sci. 119, 345-350 (2011).</li><li>Babajko, S., Jedeon, K., Houari, S., Loiodice, S., Berdal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Steroid+Axis,+a+New+Paradigm+for+Molar+Incisor+Hypomineralization+(MIH).">Disruption of Steroid Axis, a New Paradigm for Molar Incisor Hypomineralization (MIH).</a> Front Physiol. 8, 343 (2017).</li><li>Houari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asporin+and+the+mineralization+process+in+fluoride-treated+rats.">Asporin and the mineralization process in fluoride-treated rats.</a> J Bone Min Res. 29, 1446-1455 (2014).</li><li>Denbesten, P., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+fluoride+toxicity:+dental+fluorosis.">Chronic fluoride toxicity: dental fluorosis.</a> Monographs in oral science. 22, 81-96 (2011).</li><li>Kirkham, J., Robinson, C., Phull, J. K., Shore, R. C., Moxham, B. J., Berkovitz, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+rate+of+eruption+on+periodontal+ligament+glycosylaminoglycan+content+and+enamel+formation+in+the+rat+incisor.">The effect of rate of eruption on periodontal ligament glycosylaminoglycan content and enamel formation in the rat incisor.</a> Cell Tissue Res. 274, 413-419 (1993).</li><li>Lacruz, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+candidate+genes+involved+in+mineralization+of+dental+enamel+by+genome-wide+transcript+profiling.">Identification of novel candidate genes involved in mineralization of dental enamel by genome-wide transcript profiling.</a> J Cell Physiol. 227, 2264-2275 (2012).</li><li>Wen, X., Paine, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+deposition+and+ferritin+heavy+chain+(Fth)+localization+in+rodent+teeth.">Iron deposition and ferritin heavy chain (Fth) localization in rodent teeth.</a> BMC research notes. 6, 1 (2013).</li><li>Houari, S., Loiodice, S., Jedeon, K., Berdal, A., Babajko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Steroid+Receptors+in+Ameloblasts+during+Amelogenesis+in+Rat+Incisors.">Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors.</a> Front Physiol. 7, 503 (2016).</li><li>Kawano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+dental+epithelial+cell+line+(HAT-7)+and+the+cell+differentiation+dependent+on+Notch+signaling+pathway.">Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.</a> Connect Tissue Res. 43, 409-412 (2002).</li><li>Zhou, Y. L., Snead, M. 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Altered ameloblast activity and/or disrupted ameloblast proliferation, differentiation, and maturation processes lead to irreversible enamel defects and, in turn, the characterization of enamel defects may help develop understanding of the altered ameloblast activity during amelogenesis. Thus, the studies on isolated enamel organ are determinant to elucidate the pathological events leading to enamel defects whatever their origin, environmental or genetic.
This technique has originally been des...

Many developmental enamel defects may result from exposure to environmental toxicants and/or inappropriate life-style1,2,3,4. Characterization of disrupting events and molecules of amelogenesis using the presently described procedure will promote the use of resulting enamel defects as early markers of exposure to several toxicants, and may help to reconstitute the history of health of each patient during the perinatal period when enamel is synthetized1,2. Enamel synthesis can be divided into four main stages depending on ameloblast activity5. The first step regroups precursor cell and pre-ameloblast proliferation. During the second step, differentiated ameloblasts secrete enamel matrix proteins (EMPs), mainly amelogenin, enamelin and ameloblastin, which determine the thickness of the final enamel. Thus, any disruption of EMP synthesis leads to quantitative defects of enamel. After the deposition of the full enamel thickness, the maturation stage begins. During this stage, apatite crystallite growth in width and thickness allows the enamel to reach the highest mineralization ratio found in a biological tissue, with up to 96% by weight. Disrupting events that occur during the maturation stage lead to qualitative enamel defects. Finally, ameloblasts enter a phase of post-maturation, also called pigmentation in rodents, and undergo apoptosis during tooth eruption making enamel defects (if any) irreparable and irreversible, thus defects provide potential retrospective recording of ameloblast stresses. In rodents, amelogenesis follows a similar sequence of events with the particularity that their incisors are continuously growing, which makes them a suitable model to study the general process of amelogenesis. Thus, any disruption of amelogenesis results in alterations of enamel quality and/or quantity, depending on the time-window of the disrupting event. In that sense, exposure to dioxin, lead, and endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), genistein, and vinclozolin, have been shown to generate enamel hypomineralizations1,2,3,6,7,8. Asymmetric white opaque spots were identified on the incisors of rats exposed to a low-dose BPA dose during the fetal period and the first month after birth1. These enamel defects in rats, and those of human molar incisor hypomineralization (MIH), share similar clinical, structural, and biochemical characteristics. MIH is a recently described dental enamel pathology, for which the etiology still remains unclear9,10 despite many causal factors having been hypothesized9,10,11,12.
Another important enamel hypomineralization pathology due to environmental factors is dental fluorosis (DF), which is the consequence of excessive fluoride absorption (&#62;0.1 mg/kg/day)13,14. The main source of fluoride is drinking water that is either supplemented or naturally enriched with fluoride. Fluoride is also often prescribed to prevent dental caries, but the prophylactic dose is only 50% lower than the toxic one (&#8804;0.05 mg/kg/day). MIH and DF, two frequent pathologies resulting from exposure to environmental factors, may present common features that need to be characterized due to the potentiation of hypomineralizing effects of fluoride combined with other toxicants such as EDCs2 or amoxicillin15.
Micro-dissection of rat enamel organ containing ameloblasts at different differentiation stages will help to understand the mechanism of action of molecules able to disrupt ameloblast activity and cause enamel defects to be diagnosed after tooth eruption. In other words, the characterization of changes of enamel gene expression and enamel matrix composition due to environmental toxicants allows the reconstitution of the history of exposure to toxicants, and facilitates environmental safety monitoring for public health.

<table>
	<tbody>
		<tr>
			<td>Bisphenol A</td>
			<td>Sigma Aldrich, Saint Louis MO</td>
			<td>239658</td>
			<td></td>
		</tr>
		<tr>
			<td>formalin 10%</td>
			<td>Sigma-Aldrich, Saint Louis, MO</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Reagent</td>
			<td>Euromedex, France</td>
			<td>TR118</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT buffer</td>
			<td>Qiagen, Les Ulis, France</td>
			<td>74126</td>
			<td>RNeasy Protect Mini Kit</td>
		</tr>
		<tr>
			<td>Androgen receptor antibody</td>
			<td>Santa Cruz Biotechnology, Santa Cruz, CA)</td>
			<td>sc-816</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>EUOMEDEX</td>
			<td>ET330.A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride (NaF)</td>
			<td>Sigma-Aldrich, Saint Louis, MO</td>
			<td>S-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>paraplast regular</td>
			<td>Leica microsystems, Nanterre cedex,&#160;France</td>
			<td>39601006</td>
			<td>called was/parafin in the text</td>
		</tr>
		<tr>
			<td>tissue OCT</td>
			<td>VWR, Fontenay-sous-Bois, France</td>
			<td>411243</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors - Straight/8.5 cm</td>
			<td>PHYMEP , Paris, France</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Handle for Scalpel Blades - 12.5 cm</td>
			<td>PHYMEP, Paris, France</td>
			<td>10035-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Scalpel Blade</td>
			<td>PHYMEP , Paris, France</td>
			<td>10035-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Knife - Fine/Straight Tip</td>
			<td>PHYMEP , Paris, France</td>
			<td>10055-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Knife</td>
			<td>PHYMEP, Paris, France</td>
			<td>10059-15</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blades n&#176;11 Swann-Morton</td>
			<td>VWR, Fontenay-sous-Bois, France</td>
			<td>233-0024</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular lens</td>
			<td>Leica biosystems, Nanterre cedex,&#160;France</td>
			<td>MZFLIII</td>
			<td></td>
		</tr>
	</tbody>
</table>

micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Many enamel defects, such as dental fluorosis12,13, may result from environmental conditions due to excessive fluoride absorption or enamel hypomineralization similar to MIH due to exposure to some EDCs1,7,22. These developmental enamel defects may be experimentally reproduced on rats (Figure 1)<sup class="xr...

Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

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Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Operating Transverse Aortic Constriction with Absorbable Suture to Obtain Transient Myocardial Hypertrophy

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent ...